Pectin, which is mostly found in the primary cell wall and in the middle lamella, is one of the main components of the plant cell wall. Pectic substances are complex heteropolysaccharides that are mainly composed of many D-galacturonic acid residues linked by α-1,4-bonds as a backbone of pectin. The backbone can be modified by methyl-esterification or substitution with acetyl groups. Pectins can be generally classified into three groups: homogalacturonan, xylogalacturonan and rhamnogalacturonan. The last one has the most complex structure that consists of repeating rhamnose-galacturonic acid residues branched with sugars like galactose, xylose and arabinose.
Because of the complicated architecture of pectin, its complete degradation requires several different pectinolytic enzymes, also known as pectinases, to work together. These enzymes are widely produced by bacteria, yeast, fungi and plants and basically include three types: hydrolases, lyases and esterases. Among these pectinolytic enzymes, pectate lyase (also named endopolygalacturonate lyase; EC 4.2.2.2) is one of the key enzymes for depolymerization of pectin. It can randomly catalyze the α-1,4-glycosidic bond on polygalacturonic acid via transelimination mechanism and generate unsaturated oligogalacturonate products.
Pectinases have been widely used in the food and wine industries for a long time. In addition, these enzymes are extensively applied in the papermaking process, textile industry and feed manufacture. Because the industrial pectinases have high economic value in industrial applications, many studies try to find better pectinases for industrial use by either searching new genes or modifying current enzyme. Protein engineering by rational design based on structural analysis is one of the major strategies for improvement of industrial enzyme. High thermostability is one of the ideal conditions for being a good industrial enzyme. A thermostable enzyme basically has higher protein stability and better performance of enzymatic hydrolysis that means the more benefits in business.
In the present invention, the crystal structure of a pectate lyase is analyzed and the thermostability of the pectate lyase is improved by rational design.